Summary: The speed, resolution and high mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantitation of post-translational modifications (PTMs) remain a major challenge?a key limitation for many important medical applications. A key weakness with current mass spectrometry for proteomics lies in the methods used to induce fragmentation, because PTMs such as phosphorylation are among the most labile chemical bonds in proteins and are lost in complex ways by current collision-based fragmentation approaches. An alternative fragmentation methodology called electron capture dissociation (ECD) is well established to produce exceptionally clean spectra that preserve PTMs, but is currently feasible only in expensive FTICR mass spectrometers. The fundamental limitation to ECD is the difficulty of providing enough low-energy electrons to efficiently fragment peptides. We have discovered how to use carefully sculpted magnetic fields with a hot electron-producing filament to restrain large numbers of electrons in the flight path of ions. This can be adapted in any common tandem mass spectrometer without changing the existing ion optics, but our best designs can only fragment 3-5% of doubly charged trypsin-digested peptides?the most common workflow used in mass spectrometry. This low fragmentation efficiency limits sensitivity, which has proved to be the major barrier to adopting this powerful methodology by the mass spectrometry industry. The key focus of this Phase I SBIR project is determining how to increase the interaction time of ions with electrons confined to a narrow beam by the magnetic fields to prove this concept feasible. The reaction time currently is 1-2 microseconds. Our Phase I feasibility question is whether fragmentation can be effectively increased at least two-fold by transiently stopping peptide ions in the ECD cell without significant loss due to electrostatic scattering. In addition, the design must retain the sub-millisecond speed necessary to be compatible for current front-end HPLC and ion mobility separations used with mass spectrometers for complex samples. Rigorous computer simulations show these objectives can be accomplished by carefully cooling precursor ions and then transiently stopping their flight with carefully timed electrical pulses to electrostatic lenses. Proof of feasibility and validated concept demonstration (Phase II) are essential in engaging the major instrument manufacturers to further develop and commercialize our ECD technology for use in their mass spectrometer products. Success will also show how our technology can produce better fragmentation of the most challenging analytes analyzed by mass spectrometry, including lipids, glycans, and other difficult-to- fragment drugs/metabolites. The adoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic/therapeutic biomarkers with increased accuracy and greater speed, while making fewer mistaken identifications in complex biological samples.